Analysis of temperature-dependent molecular configurations

ABSTRACT

An apparatus and method for analysing temperature-dependent molecular configurations such as folding comprises a multi-channel flow-through chip ( 12 ) along which molecules to be analysed pass. A temperature gradient is maintained along the length of the chip. As molecules pass along the channels they fold or unfold, in response to the changing temperature. These changing molecular configurations are investigated by simultaneously measuring the extent to which the molecules absorb UV light, and the extent to which they fluoresce. The absorption and fluorescence information is supplied to a computer system ( 26 ) for real-time analysis.

The present invention relates generally to the analysis oftemperature-dependent molecular configurations, and particularlyalthough not exclusively to the folding of proteins, and to the bindingof proteins to other molecules.

Proteins are made up of a string of sub-units called amino acids, thesequence of which is known as the primary structure of the protein. Thisfirst level of organisation of the protein is directed by the genesequence encoding the protein, wherein a sequence of three nucleic acids(a codon) in the gene specifies the nature of the amino acid at anyparticular position. In addition to the primary structure, most proteinsalso exhibit a higher level of structural organisation. It is this threedimensional, or tertiary, structure, which allows the protein tofunction in its biological role. Many proteins in the cell exist asaggregates of two or more folded proteins, or sub-units. This level oforganisation is referred to as the quaternary structure of a protein.

Typically, proteins are made up of a number of folded domains, i.e.compact regions of folded structure. Several varieties of domains exist,including α-helices, β-sheets and β-turns.

A folded protein is held in its secondary or tertiary structure byseveral types of bonds. These include electrostatic interactions, whichoccur between the oppositely charged side chains of the amino acidsmaking up the primary structure; hydrogen bonds between amino acids;weak interactions between uncharged groups (known as van der Waalsinteractions): and disulphide bonds between cysteine amino acidresidues. Unfolding a protein by reversing these interactions is knownas denaturing the protein, back to its primary structure. This may beachieved by placing the protein in a high temperature environment, or inSDS solution.

The folding of a protein to its final, functional conformation is one ofthe last steps in protein production. It is a vital step in a complexprocess, and any error in the process can induce massive physiologicalproblems. For example, there is strong evidence to suggest that BovineSpongiform Encephalopathy (BSE) stems from a mis-folded protein (Horwichet al Cell 89 499-510 (1997)). An understanding of protein folding andstability will provide a clearer insight into the causes of disease, andtherefore will allow the development of better treatments orpreventative measures for disease.

There exist a number of techniques to study proteins. These includeX-ray crystallography ((Blundell et al Protein Crystallography London:Academic press (1976)); NMR (Clore et al Progress in NMR Spectroscopy 2343-92 (1991)); differential scanning calirometry (Blaber et al proteinstructure and Stability Florida State University (1995)); and unnaturalamino acid engineering (Mendel et al Science 256 (5065) 1798-1802(1992)).

X-ray crystallography is a preferred method for determining the threedimensional structure of proteins. However, this technique has thefundamental problem that it can only be employed when the proteins arecrystallised, and this is not always easy or even possible. This putsconstraints on the ability to study conformational changes in theprotein, changes in the folding in response to changes in environment,or interaction with other factors.

Florescence and absorption by certain optically active amino acids in aprotein have also been used to monitor conformational changes in proteinand to measure protein concentration (Chen et al Biochemistry 379976-9982 (1998)). These amino acids contain an indole chromophore whosetransitional geometry is responsible for the optical activity of theamino acid (Callis et al Chemical Physics Letters 244 53-58 (1995);Fender et al Chemical Physics Letters 262 343-348 (1996) and Fender etal Chemical Physics Letters 239 31-37 (1995)). The main amino acidscontributing to this are tryptophan and tyrosine, while phenylalanineand the disulphide bond between cysteine residues also show somefluorescence.

The problem encountered with all of the above mentioned techniques isthat they are not capable of real-time protein folding analysis. Thisimposes significant constraints on protein folding and stabilityanalysis, and therefore also on the use of proteins in the diagnosis andprevention and/or treatment of disease. Furthermore, current systemsoften reveal little about the relationship between the dynamics of thefolding and the static conformational information available.

The present invention will be useful in any method where it is desirableto analyse the folding and/or stability of a protein, and/or theinteraction of a protein with biological factors including ligands,receptors, sugars, hormones, nucleic acids and therapeutics agents.Other long chain or macromolecules may also be studied, as long as theyare able to fold or otherwise change their physical configuration inresponse to a temperature change.

According to the present invention there is provided apparatus foranalysing temperature-dependent molecular configurations, comprising:

-   -   (a) a multi-lane chip having a plurality of channels extending        along a length thereof, each channel being arranged for the        passage of molecules to be analysed, and    -   (b) heating elements associated with the chip for creating a        temperature profile along the channels, thereby exposing        molecules passing along the channels to corresponding        temperature variations.

According to a further aspect of the present invention there is provideda method of analysing temperature-dependent molecular configurations,comprising:

-   -   (a) defining a temperature profile along the length of a        channel;    -   (b) passing molecules to be analysed along the channel;    -   (c) detecting an optical characteristic of the molecules as they        pass along the channel; and    -   (d) analysing the detected optical characteristic as a function        of temperature.

To analyse the folding of a protein using the present invention, theprotein is passed along a temperature gradient as described above. Adecrease in temperature with passage of the protein will typicallyenable it to fold and adopt its secondary, tertiary or quaternarystructure. In contrast, an increase in temperature along the path of theprotein will typically cause the protein to be denatured, and unfold. Asfolding or unfolding occurs, the fluorescence emitted and light absorbedby the protein are measured, and used to generate a series of signalsillustrative of the folding pattern. The signals (also referred to asthe detected optical characteristics) produced will indicate theposition of amino acids important in folding, and the sequence offolding events which result in the final structure.

The range of the temperature across the gradient will depend upon thenature of the protein being analysed, and may be adjusted according tothe type of protein, and/or to the degree of folding or unfolding whichis desired. Typically, the temperature range will be in the region of20° C. to 200° C. The exact range for any particular molecule may bereadily determined using techniques available in the art.

The invention as described in the above paragraphs will be useful inmonitoring the quality of proteins produced recombinantly, particularlyin terms of their ability to fold correctly and therefore functionproperly, and their stability. For example, the signals generated duringthe folding of recombinant proteins (or those produced in any other wayby the hand of man) can be compared to those generated by a nativeprotein of the same type, which preferably is known to be biologicallyactive. In this way, proteins which do not share substantially the samesignal output as the native protein may be discarded, and correctlyfolded proteins may be identified. Similarly, proteins which have beendesigned to differ from the native protein in terms of structure orstability may be identified by comparative techniques using theinvention.

In the context of the present invention, a “native” protein or otherbiological factor or macromolecule is one which is found naturally invivo i.e. in the human or animal body, in plants or other life forms.

In addition, the interaction of a protein with biological factors and/orother proteins may be analysed. By folding or unfolding a protein in thepresence of a factor or protein with which it interacts, the sitescritical to the interaction may be identified. This is achieved bypassing the protein/protein or biological factor/protein complex along atemperature gradient, and monitoring the fluorescence emitted and lightabsorbed as the protein folds or unfolds. The signals generated may becompared to those generated by the same protein in the absence of thebiological factor or other protein. Comparison of the data indicates theamino acids and folded domains important to interaction, and thetemperature at which the complex breaks down.

This aspect will be particularly useful in the design of biologicalfactors, such as therapeutic agents, with the aim of reproducing,enhancing or inhibiting the interaction. In particular, once the proteinand/or biological factor domains important to interaction have beenestablished, this information can be used to design new biologicalfactors. The interaction of these with the protein, and their effect onits folding or stability can be tested using the invention. Preferably,the signals generated by the protein and native biological factorcomplex can be compared with those generated by the protein and/orcandidate biological factor, to determine whether the interaction is thesame, and if not, or how it differs.

This aspect of the invention is particularly useful in the production oftherapeutic agents. Thus, candidate therapeutic agents not having thedesired interaction can be identified by comparison of signal patternsgenerated with optimum agents or biological factors, and can bediscarded.

The present invention will also be useful in identifying variants ofproteins. A variant protein is typically one which differs from the“wild type” or reference protein in its primary structure, and may as aresult have impaired biological function and/or may serve as a markerfor disease. The wild type, or reference, protein is the functional,biologically active version.

Variations in the primary sequence may arise due to divergence in thegene sequence encoding the protein. Such divergent sites are known aspolymorphisms, and are manifested as restriction fragment lengthpolymorphisms, variable number of tandem repeats, hypervariable regions,minisatellites, di- or multi-nucleotide repeats, insertion elements andnucleotide deletions, additions or substitutions. A single nucleotidepolymorphism (SNP) is a variation in sequence at a site occupied by asingle nucleotide residue. Single nucleotide polymorphisms arise fromthe substitution, deletion or insertion of a nucleotide residue at apolymorphic site. Single nucleotide polymorphisms may result incorresponding changes to the amino acid sequence. For example,substitution of a nucleotide residue may change the codon, resulting inan amino acid change. Similarly, the deletion or insertion of threeconsecutive bases in the nucleic acid sequence may result in theinsertion or deletion of an amino acid residue.

As a result of the change in the primary sequence, a variant protein mayhave a different folding pattern, or stability, compared to the wildtype protein, or may show different interaction with biological factors.Proteins may be compared to a wild type protein in terms of theirfolding pattern, stability or interaction with biological factors bycomparing the signals generated using the present invention. In thisway, a protein may be identified as a variant or a wild type.

In addition, the invention may be used to detect SNPs in a protein. Anychange in amino acid sequence at a particular position in a protein maybe detected by comparing the signals generated by the amino acid at theposition of the wild type sequence with the signal generated by theamino acid in that position in the protein to be analysed. Polymorphismsother than SNPs which cause amino acid changes may be detected in thisway.

The invention may also be used in the correlation of polymorphisms withdisease. For example, a protein from a diseased individual may becompared with the same protein from a normal individual, and differencesin the signals generated may be used as markers for polymorphisms in theprotein, which are associated with disease. This aspect of the inventionmay be used in diagnosing an individual as having, or being susceptibleto disease. By determining the phenotype of an individual in this way,the efficacy of any treatment can be assessed.

The invention may be carried into practice in a number of ways and onespecific embodiment will now be described, by way of example, withreference to the accompanying drawings, in which:

FIG. 1 schematically illustrates and apparatus for analysing molecularfolders according to an embodiment of the present invention;

FIG. 2 is a schematic longitudinal section through the apparatus of FIG.1: and

FIG. 3 shows, again schematically, how molecules travelling along aparticular channel may be switched out under computer control.

The heart of the apparatus shown in FIG. 1 is a multi-lane chip 10, thelanes being defined by a plurality of narrow, parallel-sided open-toppedchannels 12. The width of each channel may anything between a fractionof a micrometer to hundreds of micrometers, with the depth between afraction of a micrometer to thousands of micrometers. In the preferredembodiment, the channels are about 50 micrometers wide and about 200micrometers deep. The channels could also be defined by (closed )microcapillaries within the body of the chip.

The chip may be manufactured from polydimethylsiloxane (PDMS), but otherstable, non-reactive, temperature-resistant material could also be used.The chip is of the “flow-through” type, in which a buffer containing themolecules to be analysed is continuously supplied via lead-in lines (notshown) to one end of the channels. The buffer and the molecules thenpasses along the channels and out the other end. The chip 10 ispreferably angled so that the molecules and buffer pass along thechannels simply under the force of gravity, but pumped or otherpressurised systems could also be envisaged. It would also be possibleto use electrokinetic means to transport the molecules.

Located beneath the channels 12, and at right angles thereto, are aseries of thermal heating strips 14 (only two of which are shown in FIG.1, for clarity). The temperatures of these heating strips may be variedby means of a thermal control 16, thereby introducing a temperaturegradient along the length of the channels. The thermal control 16 isable to alter the temperature gradient according to the type of analysisto be undertaken. Sometimes, for example, the temperature may bemonotonically increasing along the length of the channels (in thedirection of molecular movement), whereas in others it may bemonotonically decreasing. For some types of analysis, multi-valuedgradients may be useful—for example with low temperatures beingmaintained at the ends of the channels and a high temperature in themiddle.

As the molecules move along the channels, their physical configurationschange as they are exposed to differing temperature regimes. As is bestshown in FIG. 2, in a rising temperature regime, for example, a proteinoriginally in a folded state 17 will gradually unfold as indicated at18. Multi-valued temperature gradients, mentioned above, may be usefulin studies which seek to compare the unfolding sequence of a particularmolecule with the folding sequence. At the most basic level, a user maysimply wish to know whether, once a molecule has been unfolded byincreasing its temperature, it will naturally fold up again when thetemperature is reduced.

An array of temperature detectors such as thermocouples 20 constantlymonitors the temperature of each channel, as a function of the distance(z) along the channel. This allows a one to one mapping to be maintainedso that once a particular molecule's position (z) is known within thechannel, its temperature is then uniquely determined. So, in order tostudy how the configuration of a particular molecule varies withtemperature, we no longer need to control very closely the temperatureof a bulk sample of the molecule: instead, we simply need to watch howthe molecule changes its configuration as a function of z.

As the molecules travel along the channels they are exposed to UVillumination, from above, emanating from a UV source 22. As the UV lightpasses through the molecules, it is selectively absorbed, with theamount of absorption varying according to the physical configuration ofthe molecule. Typically, an unfolded molecule 18 will absorb less UVlight than will a folder molecule 17. The UV light passes through thePDMS chip 10 (which is substantially UV-transparent) and is detected byan underlying optical detector array 24. This may be for example aphotodiode array (PDA) or a charge coupled device (CCD). The output ofthe detector array 24 is fed to a computer system for storage andanalysis. Since the fast, real-time analysis of multiple channels isextremely processor-intensive, the computer system 26 may includehardware-based data-capture and analysis. In the preferred approach, adata-capture unit (not shown) feeds raw information to a plurality ofdigital signal processors (DSPs) controlled by a field programmable gatearray (FPGA). Of course, other data-capture and analysis systems may becontemplated. Preferably, though, the computer system 26 is capable ofcarrying out analysis in real time (i.e. as the molecules being analysedare moving along the channels).

The UV illumination from source 22 causes the molecules to fluoresce,and this fluorescence 26 is detected by means of one or more hybridphoto diodes (HPDs) 28, located above the chip. Each HPD is essentiallya multi-channel photo-multiplier tube array, which is capable ofdetecting the fluorescence from a large number of molecules at once anddetermining, for each molecule, both the channel within which it sitsand also its position (z) within the channel. z is measured sufficientlyprecisely to define the temperature narrowly enough to observe thresholdeffects and time-resolved processes. The output of the HPD, which isthus essentially a fluorescence map taken across the length and width ofthe chip, is passed for analysis to the computer system 26. Similarconsiderations apply to data-capture and analysis as were discussedabove with reference to the detector array 24.

Scattered light may be detected, instead of fluorescence. Rather thanusing an HPD, the fluorescence or scattered light may be detected bymeans of a multi-anode photo-multiplier tube.

In one embodiment (not shown) dual wavelength absorption and/orfluorescence/scattering may be measured. This allows investigation ofattraction and/or bonding between two molecules to be studied. Forexample, in a study of the bonding of sugars to proteins, theabsorption/fluorescence/scattered light may be detected at wavelengthstuned to both the protein and the sugar. Where such an approach is used,dual-wavelength IR sources are provided, along with suitabledual-wavelength detectors (not shown).

It is believed (although so far as the applicant is aware nocomprehensive research has yet been done on this) that UV absorptionmeasurements are at least partly indicative of the static properties ofthe molecules, whereas fluorescent measurements are at least partlyindicative of dynamic properties. By making both measurementssimultaneously on the same molecule, as a function of temperature,substantial additional information can be derived, over and above eithermeasurement taken on its own.

Some of the channels within the chip may be used as controls, and may befed with known types of protein or other molecules. By comparing theoutputs of the control channels and the test channels, the user caninvestigate the way in which small changes between different proteins orother molecules may affect its folding characteristics.

The precise analysis to be carried out by the computer system 26 will ofcourse depend upon the particular investigation being undertaken.Typically, however, users will be interested in the cross-correlationbetween some combination of fluorescence and absorption measurementswithin a control channel and similar measurements simultaneously beingcarried out in one or more test channels. Other possible calculations ofinterest may be the cross correlation, within a single channel, betweenthe fluorescence and the absorption measurements, as well asauto-correlation calculations. The calculations may be carried out for afixed thermal gradient, as defined by the thermal control 16, and alsofor a range of different thermal gradients. All of this is done undercontrol of the computer system 26.

A calculation output 30, such as a graphics screen or a printer,provides the user with analytical output on the analysis beingundertaken. Alternatively, or in addition, a control 32 may be providedwhich automatically controls the experimental apparatus in dependenceupon the real-time outputs. For example, the control 32 mightautomatically change the UV illumination, alter the thermal control, orchange the detection characteristics of the hybrid photo diode 28 or thedetector array 24. Also, the control 32 may provide for the automatedcollection of particular molecular samples of interest, in dependenceupon the real-time results of the analysis. One way of doing this isshown in FIG. 3.

FIG. 3 shows that the end of one of the channels 12 (in the direction offlow 31 of the molecules), is bifurcated into two output channels 32,34.Electrodes 36,38 at the entrance of the channels 32,34 allow a potentialdifference to be applied to each of the channel entrances, therebyallowing molecular samples to be selectively switched into either of thetwo channels. When a particular molecule to be collected reaches thebifircation, a high voltage is applied between the electrode 38 and anelectrode 40 at the end of the main channel, thereby diverting thesample into the channel 34. Once the sample has been collected, the highvoltage is switched from the electrode 38 to the electrode 36, therebycausing all subsequent molecules to pass down into the channel 32. Thecollected sample in the channel 34 may then be further analysed, forexample by means of a mass-spectrometer or by means of MALDI-TOF (MatrixAssisted Laser Desorption Ionisation-Time of Flight).

The switching of the voltage between the electrodes 36,38 may beeffected by the control 32, which is itself actuated by the results ofthe computer analysis carried out by the computer system 26. The control32 may also, if desired, be activated at least partly in dependence uponthe raw absorption or fluorescence information or characteristicsdefining the state of the apparatus such as the type of thermal gradientbeing applied, the time of change of any thermal gradient, the intensityof the UV illumination, the time that illumination was switched on soon. Generally, the control 32 may operate in dependence upon anyvariable of the apparatus itself, the raw data being collected, or theanalysed data. In the particular embodiment of FIG. 3, the control 32automatically acts to collect samples of particular molecules whichappear to be of some particular interest (that is, they meet somepredefined or dynamic criteria of interest based upon the real-timeresults of the analysis carried out by the computer system 26).

The system of the preferred embodiment is expected to find particularapplication in the analysis of proteins. The range of possible analysesthat can be carried out is evidently extremely broad, but they include:investigations into the thermal stability of proteins; analysis of themechanism of thermal unfolding; evaluation of the effects of mutationson thermal stability (such as SNP's in nucleic acids by which theproteins are expressed); and evaluations of the effects of environmentalconditions on molecular stability. In addition, the system may be usedto investigate how other proteins bind to molecules, and how this isaffected by temperature changes. In particular, the system may be usedto investigate how changes to a protein's thermal stability affect thecapability of small molecules to bind.

A typical analysis that could be carried out with the describedapparatus relates to the effect of different dosages of a particulardrug. For example, very high dosage regimes may cause undesirableprotein changes to occur within the patient. In order to investigatethis, protein samples would be obtained from a range of patients who arebeing treated with different amounts of the drug, and these proteinswould be compared, channel by channel, with a control channel whichcontains the corresponding “normal” protein. Such an analysis allowsvery rapid decisions to be made on the efficacy of particular drugtreatments and dosing regimes.

1. An apparatus for analysing temperature-dependent molecularconfigurations, comprising: (a) a multi-lane chip having a plurality ofchannels extending along a length thereof, each channel being arrangedfor the passage of molecules to be analysed; and, (b) heating elementsassociated with the chip for creating a temperature profile along thechannels, thereby exposing molecules passing along the channels tocorresponding temperature variations.
 2. An apparatus as claimed inclaim 1 further including temperature sensors spaced along the length ofeach channel for measuring the temperature profile.
 3. An apparatus asclaimed in claim 1 further including a detector measuring an opticalcharacteristic of the molecules as the molecules pass along thechannels.
 4. An apparatus as claimed in claim 3 in which the opticalcharacteristic is fluorescence, and in which the detector detects themolecular fluorescing in response to UV illumination as the moleculespass along the channels.
 5. An apparatus as claimed in claim 4 in whichthe detector is one of either a hybrid photodiode or a multi-anodephoto-multiplier tube.
 6. An apparatus as claimed in claim 3 in whichthe optical characteristics is UV absorption and in which the detectordetects a reduction in transmitted UV illumination, as a result ofmolecular absorption.
 7. An apparatus as claimed in claim 6 in which thedetector is one of either a charge-coupled device or a photodiode array.8. An apparatus as claimed in claim 1 further including first and seconddetectors for simultaneously measuring respective first and secondoptical characteristics of the molecules as the molecules pass along thechannels.
 9. An apparatus as claimed in claim 8 in which the firstdetector detects the molecules fluorescing in response to UVillumination and in which the second detector detects a reduction intransmitted UV illumination as a result of molecular absorption.
 10. Anapparatus as claimed in claim 3 further including a UV source forcreating UV illumination.
 11. An apparatus as claimed in claim 1 inwhich the chip is orientated so that the molecules pass along thechannels under the influence of gravity.
 12. An apparatus as claimed inclaim 1 in which the molecules are either pumped along the channels orurged along by an electric force.
 13. An apparatus as claimed in claim 3further including computer analysis means for receiving an output of thedetector(s) and arranged to analyse the output(s) as a function ofmolecular temperature.
 14. An apparatus as claimed in claim 13 furtherincluding an apparatus control arranged automatically to controloperation of the apparatus in dependence upon an output of the computeranalysis means.
 15. An apparatus as claimed in claim 14 in which theapparatus control actuates a molecule collection mechanism forautomatically collecting a sample of selected molecules from a channel.16. An apparatus as claimed in claim 15 in which the molecule collectionmechanism includes voltage switching means for switching the sample intoa side branch of the channel.
 17. An apparatus as claimed in claim 13 inwhich the computer analysis means calculates a cross-correlation betweenoutput(s) indicative of known molecules in a control channel andoutput(s) indicative of molecules for analysis in another channel. 18.An apparatus as claimed in claim 1 in which the heating elements arecontrollable to create a user-defined temperature profile along thechannels.
 19. An apparatus as claimed in claim 18 in which the heatingelements are controlled to create a monotonic increasing or decreasingtemperature profile along the channels.
 20. An apparatus as claimed inclaim 18 in which the heating elements are controlled to create amulti-valued temperature profile along the channels.
 21. A method ofanalysing temperature-dependent molecular configurations, comprising thesteps of: (a) defining a temperature profile along the length of achannel; (b) passing molecules to be analysed along the channel; (c)detecting an optical characteristic of the molecules as the moleculespass along the channel; and, (d) analysing the detected opticalcharacteristic as a function of temperature.
 22. A method as claimed inclaim 21 in which the optical characteristic is analysed as a functionof position along the channel, each position uniquely defining atemperature at that position in accordance with the profile.
 23. Amethod as claimed in claim 21 in which the optical characteristic ismolecular fluorescence.
 24. A method as claimed in claim 21 in which theoptical characteristic is the molecules' capacity for UV absorption. 25.A method as claimed in claim 21 further including the step ofsimultaneously detecting first and second optical characteristics.
 26. Amethod as claimed in claim 25 in which the first optical characteristicis molecular fluorescence and in which the second optical characteristicis the molecules' capacity for UV absorption.
 27. A method as claimed inclaim 21 further including the step of automatically collecting selectedmolecules in dependence upon a result of analysing the detected opticalcharacteristic as a function of temperature.
 28. A method as claimed inclaim 21 further including the steps of: passing control molecules alonga control channel adjacent to the channel carrying molecules to beanalysed; detecting a corresponding optical characteristic(s) of thecontrol molecules; and, comparing the optical characteristic(s) of themolecules to be analysed with the corresponding opticalcharacteristic(s).
 29. A method as claimed in claim 28 further includingthe step of calculating a cross-correlation between thecharacteristic(s) and the corresponding characteristic(s).
 30. A methodas claimed in claim 21 in which the molecules to be analysed aremacro-molecules.
 31. A method as claimed in claim 21 in which themolecules to be analysed are proteins.
 32. A method as claimed in claim21 comprising the step of analysing molecular folding as a function oftemperature.
 33. A method as claimed in claim 21 comprising analysingmolecular bonding as a function of temperature.
 34. A method as claimedin claim 21 comprising the step of analysing molecular stability as afunction of temperature.
 35. A method as claimed in claim 21 furtherincluding the step of using the detected optical characteristic(s) todetermine either a folding pattern or a sequence of folding events ofthe molecule.
 36. A method as claimed in claim 21 further including thestep of using the detected optical characteristic(s) to identifyresidues of the molecule involved in folding.
 37. A method according toclaim 36 wherein the molecule is a protein and the residues are aminoacids.
 38. A method as claimed in claim 21 wherein the molecule to beanalysed is either a protein/biological factor or protein/proteincomplex.
 39. A method according to claim 38 wherein the biologicalfactor is a sugar, hormone, ligand, receptor or nucleic acid.
 40. Amethod as claimed in claim 38 further including the step of determiningthe domains necessary for interaction of a protein with a biologicalfactor or protein.
 41. A method as claimed in claim 40 further includingthe step of designing therapeutic agents therefrom.
 42. A method asclaimed in claim 41 further including the steps of: passing the nativeprotein/biological factor or protein/protein complex along a controlchannel adjacent to the channel carrying the protein/candidatetherapeutic agent complex to be analysed; detecting a correspondingoptical characteristic(s) of the native protein/biological factor orprotein/protein complex; and, comparing the optical characteristic(s) ofthe protein/candidate therapeutic agent complex to be analysed with thecorresponding optical characteristic(s).
 43. A method as claimed inclaim 28 further including the step of analysing a recombinant protein,wherein the control molecule is the native protein and the molecule tobe analysed is the recombinant protein.
 44. A method as claimed in claim21 further including the steps of: comparing the detected opticalcharacteristic(s) of a wild type protein with the detected opticalcharacteristic(s) of a candidate protein; and, identifying apolymorphism in a protein.
 45. A method as claimed in claim 44 whereinthe polymorphism is a SNP.
 46. A method as claimed in claim 21 furtherincluding the steps of: comparing the detected optical characteristic(s)of the protein with those of a wild type or a variant protein; and,determining whether a protein is either a wild type or a variant.
 47. Amethod as claimed in claim 21 further includingv the steps of: comparingthe detected optical characteristic(s) of a molecule from a non-diseasedindividual with detected optical characteristic(s) of the correspondingmolecule from a diseased individual; and, identifying polymorphismsassociated with disease.
 48. A method as claimed in claim 21 furtherincluding the steps of: comparing the detected optical characteristic(s)of a molecule from an individual with the detected opticalcharacteristic(s) of the form of the molecule known to be associatedwith a disease; and, diagnosing an individual as having, or beingsusceptible to, disease.
 49. A method as claimed in claim 48 wherein themolecule to be analysed is from a sample removed from a human or animalbody.
 50. An apparatus as claimed in claim 2 further including first andsecond detectors measuring respective first and second opticalcharacteristics of the molecules.
 51. An apparatus as claimed in claim50 in which the first and second detectors are receptive to light offirst and second wavelengths, respectively.